TANK-binding kinase 1 (TBK1/NAK/T2K) and I-kappaB Kinase (IKK-i/IKK-epsilon) play important roles in the regulation of interferon (IFN)-inducible genes during the immune response to bacterial and viral infections. Cell stimulation with ssRNA virus, dsDNA virus or gram-negative bacteria leads to activation of TBK1 or IKK-i, which in turn phosphorylates the transcription factors, IFN-regulatory factor (IRF) 3 and IRF7, promoting their translocation in the nucleus. To understand the molecular basis of activation of TBK1, we analyzed the sequence of TBK1 and IKK-i and identified a ubiquitin-like domain (ULD) adjacent to their kinase domains. Deletion or mutations of the ULD in TBK1 or IKK-i impaired activation of respective kinases, failed to induce IRF3 phosphorylation and nuclear localization and to activate IFN-beta or RANTES promoters. The importance of the ULD of TBK1 in LPS- or poly(I:C)-stimulated IFN-beta production was demonstrated by reconstitution experiments in TBK1-IKK-i-deficient cells. We propose that the ULD is a regulatory component of the TBK1/IKK-i kinases involved in the control of the kinase activation, substrate presentation and downstream signaling pathways.